Biology subjects

Janssen, A.

Publications and source records attributed to Janssen, A..

16 recordsLinked to original sources

bioRxiv2026

Mapping subcellular H2O2 dynamics reveals tissue specific redox patterns in Drosophila

Redox signalling regulates development, tissue homeostasis, and organismal health. Hydrogen peroxide (H2O2) is a major signalling form of reactive oxygen species (ROS) that modulates protein activity through oxidation of redox-sensitive cysteines that is reversed by cellular reducing systems. Since H2O2 production, scavenging and reduction are spatially restricted, signalling specificity is strongly influenced by subcellular localisation. However, subcellular H2O2 dynamics in animal tissues remain poorly understood. To address this, we generated and validated Drosophila melanogaster lines expressing the ultrasensitive, ultrafast ratiometric H2O2 biosensor HyPer7 targeted to mitochondria, nucleus, cytosol, or plasma membrane. With its highly conserved metabolic and signalling pathways, tractable lifespan, and powerful genetic toolkit, Drosophila is an ideal model for studying redox biology. These new 'FlyPer' lines enable tissue-specific HyPer7 expression and high-resolution measurement of subcellular, in vivo H2O2 dynamics throughout the lifespan. Using FlyPer, we detected compartment-specific H2O2 dynamics during oxidative stress, ageing, wing disc development and embryogenesis, uncovering unexpected patterns of spatially and temporally regulated oxidation throughout the organism. Together, these findings establish FlyPer as a valuable toolkit for in vivo redox biology and suggest that compartmentalised redox dynamics are a fundamental yet still poorly understood layer of developmental programming.

cell biology↗

bioRxiv2026

Rapid removal of nuclear aggregates via proteasome- and VCP-dependent disaggregation

Cells use multiple protein quality control (PQC) mechanisms to counteract the toxic effects of protein aggregation caused by cellular stress, ageing or disease. While it is known that PQC mechanisms differ between cellular compartments, studying such differences has remained challenging. Previously, we developed an assay to study cytosolic PQC using aggregates formed through chemically-induced dimerization (termed PIMs, particles induced by multimerization). Here, we introduce nuclear PIMs as a tool to study nuclear quality control. Using high-resolution and high-throughput imaging, we show that nuclear aggregate removal depends on the proteasome and the unfoldase VCP, but not on Hsp70. Strikingly, following dissolution many PIM subunits were exported to the cytosol via exportin-1-dependent shuttling, indicating that disaggregation and resolubilization dominated over degradation. Proteasomal disaggregation was confirmed using live-cell turnover experiments. Together, these findings reveal a mechanism in which the proteasome and VCP disaggregate, rather than degrade, nuclear protein aggregates, with the resulting subunits subsequently cleared via cytosolic aggrephagy.

cell biology↗

bioRxiv2026

Platform-Imprinted Transcriptional and Clonal Remodeling of αβ and γδT Cells After Allogeneic Transplantation

Immune reconstitution after allogeneic hematopoietic stem cell transplantation is influenced by graft-composition and viral reactivation, but the combined long-term impact on {beta} and {gamma}{delta}T cells remains unclear. We analyzed a cohort of 213 patients receiving either {beta}T cell-depleted grafts (n=146; graft engineering that removes donor {beta}T cells) or T cell-replete grafts (n=67; containing donor T cells). Longitudinal immune phenotyping was integrated with bulk and single-cell TCR repertoire and transcriptomic profiling. CMV reactivation was associated with expansion of CD8+ {beta}T cells across both transplant types and with numerical dominance of V{delta}2- {gamma}{delta}T cells specifically in {beta}T cell-depleted recipients. V{delta}2- {gamma}{delta}T cells underwent early polyclonal expansion followed by repertoire focusing, independent of CMV, whereas {beta}T cells remained clonally restricted. Reduced early V{delta}2+ {gamma}{delta}TCR diversity was associated with EBV reactivation. Single-cell and TCR tracking analyses revealed long-term persistence of donor-derived V{delta}2+ {gamma}{delta}TCRs, whereas V{delta}1+ {gamma}{delta} and {beta}T cell repertoires were predominantly rebuilt de novo. Despite de novo rebuilding, {beta}TCR repertoire diversity diverged by platform at one year: {beta}T cell-depleted recipients exhibited marked (hyper)expansion of {beta}TCR clonotypes and lower diversity than T cell-replete recipients, indicating a durable imprint of graft engineering on {beta}TCR-clonality. Transcriptomic profiling showed that post-transplant T cells predominantly adopted effector programs, with platform-dependent polarization toward cytotoxic signatures in {beta}T cell-depleted recipients and toward AREG-associated tissue-repair signatures in T cell-replete recipients, consistent with wound-healing functions. In conclusion, transplantation platforms imprint durable clonal and transcriptional remodeling of {beta} and {gamma}{delta}T cells, while viral reactivation primarily amplifies expansion without fundamentally reshaping repertoire architecture. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/704768v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@88f136org.highwire.dtl.DTLVardef@944f13org.highwire.dtl.DTLVardef@d3835corg.highwire.dtl.DTLVardef@5548ef_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

Transcriptomic profiling of human γδ T cells reveals non-linear immune aging characterized by childhood transitions and relative stability in adulthood

{gamma}{delta} T cells are one of the first T cell subsets developing in early ontogeny and show various effector functions in immune homeostasis and response in the young and the old. However, their maturation trajectories from infancy to children, adults and elderly have not been systematically defined. Here, we generated a single-cell transcriptome atlas of 106,711 {gamma}{delta} T cells from 223 individuals spanning infancy to old age. Our analysis reveals that {gamma}{delta} T cell aging is non-linear, characterized by pronounced childhood transitions followed by relative stability throughout adulthood despite marked inter-individual variability. In childhood, changes from developmental and mitochondrial programs toward cytotoxicity and inflammaging were evident. This includes maturation trajectories from GZMK intermediates to GZMB+Perforin+ effectors at both RNA and protein levels. Taken together, our study delineates the aging trajectories of human {gamma}{delta} T cells, establishes {gamma}{delta} T cells as a cellular paradigm of non-linear immune aging, and provides a comprehensive resource for investigating {gamma}{delta} T cell biology across the human lifespan.

immunology↗

bioRxiv2025

Non-cell-autonomous mechanisms of tumor initiation and relapse by chromosomal instability

Chromosomal instability (CIN) is a hallmark of cancer, and a primary cause of genetic heterogeneity in tumors. Depending on the degree of CIN and the affected tissue, CIN can promote or suppress tumor formation, and high CIN induction has been proposed as a therapeutic strategy. How CIN achieves these effects is unclear. Here we use a conditional mouse model of graded CIN in combination with longitudinal monitoring of DMBA/TPA-initiated skin tumors to show that low CIN increases the frequency of skin tumor initiation, while higher CIN accelerates onset and growth rates. Strikingly, gene recombination analysis of the tumors reveals that upon high CIN induction the fast-growing tumors originate from rare low CIN cells, suggesting a strong non-cell-autonomous effect of high CIN. Gene expression analysis and immunohistochemistry show that high CIN causes epidermal hyperplasia, immune evasion and a regenerative response that stimulates low CIN tumor growth beyond what is achieved by induction of low CIN alone. Such cell-extrinsic effects may be a common mechanism of tumor formation by CIN, as we observe it also in CIN-induced tumors of the intestine, breast and mesentery. When high CIN is induced in established skin tumors, mimicking CIN-based therapy, tumors regress but relapse quickly. Relapsed tumors, too, arose from rare low CIN cells. Our findings have implications for our understanding of the contributions of CIN to cancer initiation and progression and give caution to the rationale for CIN therapies. Significance statementChromosomal instability (CIN) is a source of genetic diversity in cancer. Different degrees of CIN can be highly oncogenic or tumor suppressive, the latter being a rationale for CIN-based therapies. Using a mouse model for precise control of CIN levels, we show that low CIN promotes tumor initiation, while moderate CIN accelerates both initiation and tumor growth. Surprisingly, moderate CIN acts through its effects on the surrounding tissue rather than directly within tumor cells, by triggering a regenerative response accompanied by immune evasion. When very high CIN is induced in existing tumors, tumors initially regress but rapidly relapse. These findings inform on the contributions of CIN to cancer initiation and progression and give caution to the rationale for CIN therapies.

cancer biology↗

bioRxiv2024

Varying recombination landscapes between individuals are driven by polymorphic transposable elements

AbstractsMeiotic recombination is a prominent force shaping genome evolution, and understanding the causes for varying recombination landscapes within and between species has remained a central, though challenging, question. Recombination rates are widely observed to negatively associate with the abundance of transposable elements (TEs), selfish genetic elements that move between genomic locations. While such associations are usually interpreted as recombination influencing the efficacy of selection at removing TEs, accumulating findings suggest that TEs could instead be the cause rather than the consequence. To test this prediction, we formally investigated the influence of polymorphic, putatively active TEs on recombination rates. We developed and benchmarked a novel approach that uses PacBio long-read sequencing to efficiently, accurately, and cost-effectively identify crossovers (COs), a key recombination product, among large numbers of pooled recombinant individuals. By applying this approach to Drosophila strains with distinct TE insertion profiles, we found that polymorphic TEs, especially RNA-based TEs and TEs with local enrichment of repressive marks, reduce the occurrence of COs. Such an effect leads to different CO frequencies between homologous sequences with and without TEs, contributing to varying CO maps between individuals. The suppressive effect of TEs on CO is further supported by two orthogonal approaches-analyzing the distributions of COs in panels of recombinant inbred lines in relation to TE polymorphism and applying marker-assisted estimations of CO frequencies to isogenic strains with and without transgenically inserted TEs. Our investigations reveal how the constantly changing mobilome can actively modify recombination landscapes, shaping genome evolution within and between species.

genomics↗

bioRxiv2024

A human-specific, concerted repression of microcephaly genes contributes to radiation-induced growth defects in forebrain organoids

AbstractPrenatal radiation-induced DNA damage poses a significant threat to normal brain development, resulting in microcephaly which primarily affects the cerebral cortex. It is unclear which molecular mechanisms are at the basis of this defect in humans as the few mechanistic studies performed so far were done in animals. Here, we leveraged human embryonic stem cell- derived forebrain organoids as a model for human corticogenesis. Organoids were X-irradiated with a moderate and a high dose at different time points, representing very early and mid corticogenesis. Irradiation caused a dose- and developmental-timing-dependent reduction in organoid size, which was more prominent in developmentally younger organoids. This coincided with a dose-dependent canonical p53-DREAM-dependent DNA damage response (DDR), consisting of cell cycle arrest, DNA repair and apoptosis. The DDR was delayed and less pronounced in the older organoids. Besides the DDR, we observed radiation-induced premature differentiation of neural progenitors and changes in metabolism. Importantly, our transcriptomic analysis furthermore demonstrated a concerted p53-E2F4-dependent repression of primary microcephaly genes. We found that this was a human-specific feature, as it was not observed in mouse embryonic brains or primary mouse neural progenitor cells. Thus, human forebrain organoids are an excellent model to investigate prenatal DNA damage-induced microcephaly and to uncover potentially targetable human-specific pathways.

developmental biology↗

bioRxiv2024

Protective function of ex vivo expanded CD8 T cells in a mouse model of adoptive therapy for cytomegalovirus infection depends on integrin beta 1 but not CXCR3, CTLA4, or PD-1 expression

The adoptive transfer of virus-specific T cells (VSTs) represents a therapeutic option for viral infection treatment in immunocompromised patients. Before administration, ex vivo culture enables VST expansion. However, it is unclear how ex vivo expansion affects the circulation, homing, and intra-tissue migration of administered VSTs. We established a model of VST immunotherapy of acute cytomegalovirus infection using adoptive transfer of ex vivo expanded OT-I CD8 T cells (recognizing SIINFEKL peptide) into Rag2-/- mice infected with murine cytomegalovirus (MCMV) encoding for the SIINFEKL peptide. Ex vivo expansion induced an effector T cell phenotype and affected the expression of integrins and chemokine receptors. CRISPR/Cas9-mediated gene deletions enabled us to address the role of selected genes in the homing of VSTs following intravenous administration. We found that deletion of Itgb1, encoding for integrin beta 1, prevented OT-I cells from entering infected organs and drastically reduced their number in blood, suggesting that adoptively transferred VSTs primarily expand in the infected tissues. In contrast, Cxcr3-/- OT-I cells provided equal protection as their Cxcr3+/+ counterparts, indicating that this chemokine receptor does not contribute to VST entry into infected organs. Further, Pdcd1 and Ctla4 deletion did not impair the transferred OT-I cells ability to protect mice from MCMV, arguing against quick exhaustion of VSTs with an effector T cell phenotype. Together, these data indicate that ex vivo expansion affects migration and activation properties of VSTs and suggest that future clinical evaluation of adoptive T cell therapy efficacy should include homing molecule expression assessment.

immunology↗

bioRxiv2024

DNA density is a better indicator of a nuclear bleb than lamin B loss

Nuclear blebs are herniations of the nucleus that occur in diseased nuclei that cause nuclear rupture leading to cellular dysfunction. Chromatin and lamins are two of the major structural components of the nucleus that maintain its shape and function, but their relative roles in nuclear blebbing remain elusive. Lamin B is reported to be lost in blebs by qualitative data while quantitative studies reveal a spectrum of lamin B levels in nuclear blebs dependent on perturbation and cell type. Chromatin has been reported to be decreased or de-compacted in nuclear blebs, but again the data are not conclusive. To determine the composition of nuclear blebs, we compared the immunofluorescence intensity of lamin B and DNA in the main nucleus body and nuclear bleb across cell types and perturbations. Lamin B nuclear bleb levels varied drastically across MEF wild type and chromatin or lamins perturbations, HCT116 lamin B1-GFP imaging, and human disease model cells of progeria and prostate cancer. However, DNA concentration was consistently decreased to about half that of the main nucleus body across all measured conditions. Using Partial Wave Spectroscopic (PWS) microscopy to measure chromatin density in the nuclear bleb vs body we find similar results that DNA is consistently less dense in nuclear blebs. Thus, our data spanning many different cell types and perturbations supports that decreased DNA is a better marker of a nuclear bleb than lamin B levels that vary widely.

cell biology↗

bioRxiv2023

Double-strand breaks in facultative heterochromatin require specific movements and chromatin changes for efficient repair

DNA double-strand breaks (DSBs) must be properly repaired within diverse chromatin domains to maintain genome stability. Whereas euchromatin has an open structure and is associated with active transcription, facultative heterochromatin is essential to silence developmental genes and forms compact nuclear condensates, called polycomb bodies. Whether the specific chromatin properties of facultative heterochromatin require distinct DSB repair mechanisms remains unknown. Here, we integrate single DSB systems in euchromatin and facultative heterochromatin in Drosophila melanogaster and find that facultative heterochromatic DSBs rapidly move outside polycomb bodies. These DSB movements coincide with a break-proximal reduction in the canonical heterochromatin mark histone H3 Lysine 27 trimethylation (H3K27me3). We demonstrate that DSB movement and loss of H3K27me3 at heterochromatic DSBs both depend on the histone demethylase dUtx. Moreover, loss of dUtx specifically disrupts completion of homologous recombination at heterochromatic DSBs. We conclude that DSBs in facultative heterochromatin require dUtx-mediated loss of H3K27me3 to promote DSB movement and repair.

molecular biology↗

bioRxiv2023

DNA Double-Strand Break Movement in Heterochromatin Depends on the Drosophila Histone Acetyltransferase Gcn5.

Cells employ diverse strategies to repair double-strand breaks (DSBs), a dangerous form of DNA damage that threatens genome integrity. Eukaryotic nuclei consist of different chromatin environments, each displaying distinct molecular and biophysical properties that can significantly influence the DSB repair process. Specifically, DSBs arising in the compact and silenced heterochromatin domains have been found to move to the heterochromatin periphery in mouse and Drosophila to prevent aberrant recombination events. However, it is poorly understood how chromatin components, such as histone post-translational modifications, contribute to these DSB movements within heterochromatin. Using locus-specific DSB induction in Drosophila tissues and cultured cells, we identify that histone H3 lysine 9 acetylation (H3K9ac) is enriched at DSBs in heterochromatin but not euchromatin. We find that this enrichment is mediated by the histone acetyltransferase dGcn5, which rapidly localizes to heterochromatic DSBs. Moreover, we demonstrate that in the absence of dGcn5, heterochromatic DSBs display impaired recruitment of the SUMO E3 ligase Nse2/Qjt and fail to relocate to the heterochromatin periphery to complete repair. In summary, our results reveal a previously unidentified role for dGcn5 and H3K9ac in heterochromatin DSB repair and underscore the importance of differential chromatin responses at heterochromatic and euchromatic DSBs to promote safe repair.

cell biology↗

bioRxiv2023

Adult thymus-derived cMaf+ RORγt+ γδ T cells lack Scart2 chromatin accessibility and do not reach periphery

T cell receptor (TCR) V{gamma}4+ expressing {gamma}{delta} T cells can be divided into IFN-{gamma} and IL-17-producing effector T cell subsets. A bias towards {gamma}{delta}17 effector fate decisions is observed during early ontogeny. In contrast, the existence of V{gamma}4+ {gamma}{delta}17 cells derived from adult thymus is still controversial. In the present work, we used a mouse model where T cells are exclusive generated within an adult thymus. Additionally, we employed single-cell chromatin state analysis from thymocytes of normal mice. A small, but considerable population of immature Cd24+ Gzma+ V{gamma}4 cells was found that exhibit molecular programs of {gamma}{delta}17 cells. These adult thymus-derived immature Cd24a+ cMaf+ V{gamma}4 cells secrete small amounts of IL-17A and IL-17F. Interestingly, do not reach the periphery under steady-state conditions. Furthermore, de novo generated {gamma}{delta}17-like cells from adult thymus lack transcriptional activity of the Scart2 encoding gene, suggesting that Scart2 is a distinct trait of fetal {gamma}{delta} T cell precursors. Together, this study provides valuable insights into developmental traits of V{gamma}4 cells during adulthood and raises the question on signals suppressing the full maturation and/or thymic export of {gamma}{delta}17-like cells within the adult thymus. HighlightsO_LITranscriptional and epigenetic profiling identifies developmental plasticity of Gzma+ Cd24a+ V{gamma}4 cells in adult thymus. C_LIO_LIThymic c-Maf+ and ROR{gamma}t+ V{gamma}4 T cells can be generated during adulthood, but do not reach the periphery under steady-state conditions. C_LIO_LIInnate CD44highCD45RBneg {gamma}{delta}17 cells are completely absent upon induction of T cell development during adulthood. C_LIO_LIScart2 expression might be a key molecule to track developmental traits of fetal-derived {gamma}{delta}17 cell precursors. C_LI

immunology↗

bioRxiv2022

The soybean rust pathogen Phakopsora pachyrhizi displays transposable element proliferation that correlates with broad host-range adaptation on legumes

Asian soybean rust, caused by Phakopsora pachyrhizi, is one of the worlds most economically damaging agricultural diseases. Despite P. pachyrhizis impact, the exceptional size and complexity of its genome prevented generation of an accurate genome assembly. We simultaneously sequenced three P. pachyrhizi genomes uncovering a genome up to 1.25 Gb comprising two haplotypes with a transposable element (TE) content of ~93%. The proliferation of TEs within the genome occurred in several bursts and correlates with the radiation and speciation of the legumes. We present data of clear de-repression of TEs that mirrors expression of virulence-related candidate effectors. We can see a unique expansion in amino acid metabolism for this fungus. Our data shows that TEs play a dominant role in P. pachyrhizis genome and have a key impact on various processes such as host range adaptation, stress responses and genetic plasticity of the genome.

microbiology↗

bioRxiv2021

Tumor-reactivity of d2 negative gdT cells and the role of the gdT cell receptor

{beta}T cells engineered to express a defined {gamma}{delta}TCR (TEG) to attack cancer cells have shown great promise when using a {gamma}9{delta}2TCR to redirect {beta}T cells (1-9). Reports by us (1-9) and recent reports by others (10, 11) support the key role of the {gamma}9{delta}2TCR in cancer recognition. We further emphasized the crucial role of the {delta}TCR chain and that differences in CDR3 sequences of the {delta}TCR chain modulates functional avidity of TEGs (2, 8). We and others demonstrated that also {delta}2 negative {gamma}{delta}TCRs are able to redirect {beta}T cells towards different tumor cell lines (12-15). However, some studies suggest that {delta}2 negative {gamma}{delta}TCRs play a minor role in the tumor recognition by {delta}2 negative {gamma}{delta}T cells (16, 17). In addition for both modes of action for tumor-recognition, {delta}2 negative {gamma}{delta}TCR-dependent and -independent, it has been suggested that CMV infection is not only a major driver of {delta}2 negative {gamma}{delta}T cell expansion (18) but also induces tumor-cross reactive {delta}2 negative {gamma}{delta}T cells (19-21). Therefore, we aimed to systematically explore frequencies of tumor reactive {delta}2 negative {gamma}{delta}T cells in naive repertoires (cord blood) and patients with or without CMV infection and examined the potential role the parental {delta}2 negative {gamma}{delta}TCR in anti-tumor reactivity of selected clones. We observed that approximately 30% of all tested clones were tumor-reactive, though no differences were observed between different sources. Surprisingly, none of the so far tested {gamma}{delta}TCR did mediate strong anti-tumor reactivity of the parental clones. Though numbers of tested TCR sequences are still low, our data imply that tumor-reactivity of {delta}2 negative {gamma}{delta}T cells is frequently not mediated by the {delta}2 negative {gamma}{delta}TCR alone.

immunology↗

bioRxiv2019

Degree and site of chromosomal instability define its oncogenic potential

Most human cancers are aneuploid, due to a chromosomal instability (CIN) phenotype. Despite being hallmarks of cancer, however, the roles of CIN and aneuploidy in tumor formation have not unequivocally emerged from animal studies and are thus still unclear. CIN can both promote and suppress tumorigenesis, but variances in mechanisms and timings of CIN induction in different oncogenic backgrounds and associated tissues limit interpretation of the contributions of CIN. Using a novel conditional mouse model for diverse degrees of CIN, we find that a particular range is sufficient to drive very early onset spontaneous adenoma formation in the intestine, showing that CIN can act as a much more potent oncogenic driver than was previously reported. In mice predisposed to intestinal cancer (ApcMin/+), moderate but not low CIN causes a remarkable increase in adenoma burden in the entire intestinal tract, especially in the distal colon, more closely modelling human disease. Strikingly, high levels of CIN promote adenoma formation in the distal colon even more than moderate CIN does, but have no effect in the small intestine. Our results thus show that CIN can be potently oncogenic, but that certain levels of CIN can have contrasting effects in distinct tissues.

cancer biology↗

bioRxiv2018

Timely double-strand break repair and pathway choice in pericentromeric heterochromatin depend on the histone demethylase dKDM4A

Introduction Introduction Results Discussion Materials Methods References One of the most harmful DNA lesions is a double-strand break (DSB), whose improper repair can lead to formation of aberrant chromosomes linked to cancer and developmental diseases 1. At DSBs, the severed strands of the DNA helix are repaired by a variety of mechanisms. The two major DSB repair pathways are Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). NHEJ repairs DNA by ligating both ends of the DSB together, often resulting in small insertions and deletions at the break site. HR repair involves more extensive processing of the DSB site, in which 5 to 3 end-resection of the DSB ends results in a single-stranded DNA sequence that invades and perfectly copies homologous sequences to repair the DS ...

cell biology↗